Several publications and patents are referenced in this application to describe the state of the art to which the invention pertains. Each of these publications or patents is incorporated by reference herein.
RNA viruses and retroviruses use viral encoded polymerases which have a low fidelity, thereby increasing the number of mutations that occur during replication of the viral genome. This low fidelity results in a virus population that contains a large number of variants. The large variation enables these viruses to rapidly evolve to adapt to a changing environment, such as a reactive immune system, or to lose attenuating mutations introduced to limit replication of the virus in the host.
The RNA-dependent RNA polymerase (RDRP), has an error rate of about 10−4 (i.e., one error is introduced per every 10,000 nucleotides). Thus, each time the average RNA virus genome (ranging in size from 7,400 to ˜20,000 bases) is replicated in either the positive or negative strands, at least one new random error is introduced. Reverse transcriptase (RT) has a similar error rate.
A good example of the negative impact of such a mutation rate can be observed in the Sabin poliovirus vaccine, which is a live, attenuated virus carrying a single primary attenuating mutation in the 5′ non-translated region. Within 3–5 days post-vaccination in children, the poliovirus shed in the stool has reverted to a virulent genome and the major attenuating mutation site has changed back to wild-type.
The main factor contributing to high mutation rates in these viruses is the absence or low efficiency of proofreading or repair activities associated with RDRPs. Structurally, the RDRPs are similar to one another (for instance, HIV reverse transcriptase and poliovirus RNA polymerase are quite similar). The poliovirus RNA polymerase is shaped like a hand that is making an effort to hold a cup or glass, fingers somewhat together and curled, thumbs apart, with a palm in between (see, e.g., Hansen, J. L., A. M. Long and S. C. Schulz (1997); Structure 5: 1109–1122). The palm region contains the active site for both the poliovirus and HIV polymerases, and the region contains similar motifs in both enzymes. In HIV strains from patients who no longer respond to nucleotide therapy, mutants have been isolated that (a) have mutations in the palm region, (b) show many-fold (e.g., 3–49 fold) better fidelity, and (c) replicate the genome at a reduced rate.
Clearly, the aforementioned high reversion rate in RNA viruses and other viruses utilizing RDRPs or RTs, which is caused by their low-fidelity polymerases, is detrimental to their utility as vaccines or as vectors for delivery of other genes of interest. Thus, the field of viral vaccines and vectors would be vastly improved through the development of improved vectors encoding RDRPs or RTs with greater replicative fidelity, such that attenuations introduced into the viral genomes are retained for longer periods of time, without reversion to wild-type virus.